This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cell polarity is essential for virtually all aspects of cell behavior including cell morphology, cell motility, and cell identity arising from asymmetric divisions. Cell polarity has been most extensively studied in genetic systems, but while this has led to the discovery of many regulators, many more remain to be identified. Non-canonical Wnt signaling (i.e. [unreadable]-catenin independent) controls many instances of cell polarity such as during cell division or in establishing directional morphology of neurons. Many genes involved in cell polarity have been identified through genetic screens including cell receptors, cytoskeleton regulators, and Wnt signaling molecules. Yet it is not clear how these genes work together mechanistically to regulate cell polarity. Our goal is to integrate biochemical approaches with EM tomography in order to build a mechanistic model of how cell polarity is controlled. Wnt regulates polarized receptor localization in many developmental systems, but whether this occurs early or late during cell polarization is unclear. We developed an unorthodox strategy of imaging cells after acute stimulation of Wnt signaling using known concentrations of ligand for controlled periods of time, and discovered an unprecedented mechanism to explain how cell polarity and directional cell movement are controlled in response to signaling through Wnt5a. These were applied to melanoma cells, where Wnt5a has been shown to promote cell invasion (1). By treating melanoma cell lines with purified Wnt5a, we discovered a novel polarized structure induced by Wnt5a (2). The complex is composed of an IgG-family cell adhesion molecule (melanoma cell adhesion molecule, or "MCAM"), F-actin, and myosin IIB. This unique complex, which we refer to as the Wnt-Receptor-Actin-Myosin (WRAM) complex", forms within 30 minutes of exposure to Wnt5a and reflects a more widespread polarization of the cell. Wnt5a induces a polarized localization of the WRAM complex at the cell posterior, and its formation leads to rapid retraction of membrane, leading to cell movement towards the anterior direction. Thus, formation of the WRAM complex controls directional cell movement, by regulating posterior membrane retraction. In the presence of a chemotactic gradient, the WRAM complex orients distally with respect to Golgi, indicating cell polarization by Wnt5a in the context of a chemotactic cue. This novel response to acute Wnt5a exposure allows the study of early events which initiate cell polarization, not easily accomplished by classic genetic approaches. Correlative electron microscopy (EM) and EM tomography are being used to map the changing structure of the cell during WRAM complex formation. We are imaging live cells expressing MCAM-GFP l and have frozen by rapid freezing at different times after Wnt5a treatment, capturing different steps in WRAM complex formation. This will make it possible to correlate changes in ultrastructure with changes in the WRAM complex. Correlative EM will allow us to observe MVBs at specific steps distinguished by internalization, membrane trafficking, and interaction with cytoskeletal and plasma membrane proteins.